Comparison of detection methods and follow-up study on the tyrosine kinase inhibitors therapy in non-small cell lung cancer patients with ROS1 fusion rearrangement

Abstract

Background: The screening of ROS proto-oncogene 1, receptor tyrosine kinase(ROS1) fusion rearrangement might be potentially beneficial for an effective therapy against non-small cell lung cancer (NSCLC). However, the three main ROS1 rearrangement detection methods have limitations, and no routine protocol for the detection of ROS1 rearrangement in NSCLC is available. In this study, our aims were to compare immunohistochemistry (IHC), fluorescent in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (qRT-PCR) in their ability to detect ROS1 rearrangement in NSCLC, and discuss the clinical characteristics and histopathology of the patients with ROS1 rearrangement. Moreover, the effects of tyrosine kinase inhibitors (TKIs) therapy on the patients with ROS1 rearrangement and advanced stage disease (III b-IV) were investigated.

Methods: Patients with a previously diagnosed NSCLC were recruited in this study from November 2013 to October 2015. IHC was performed using the D4D6 monoclonal antibody (mAb) in an automatic IHC instrument, while FISH and qRT-PCR were carried out to confirm the IHC results. FISH and qRT-PCR positive cases underwent direct sequencing. After detection, patients with advanced ROS1 rearranged NSCLC had received TKI therapy.

Results: Two hundred and thirty-eight patients were included in this study. ROS1 rearrangement was detected in 10 patients. The concordant rate of FISH and qRT-PCR results was 100 %, while in the FISH and IHC results high congruence was present when IHC showed a diffusely (≥60 % tumor cells) 2-3+ cytoplasmic reactivity pattern. Patients harboring ROS1 rearrangement were mostly young (8/10), females (7/10) and non-smokers (7/10) with adenocarcinoma (10/10) and acinar pattern. Most of their tumor were in intermediate grade (6/8). Among these 10 patients, three of them in stage IV with ROS1 rearrangement gained benefits from ROS1 TKI therapy.

Conclusions: IHC, FISH and qRT-PCR can reliably detect ROS1 rearrangement in NSCLC, while IHC can be used as a preliminary screening tool. These results supported the efficacy of ROS1 TKI therapy in treating advanced NSCLC patients with ROS1 rearrangement.

Keywords: Fluorescent in situ hybridization; Immunohistochemistry; Non-small cell lung cancer; Quantitative real-time polymerase chain reaction; ROS1; Tyrosine kinase inhibitors.

Fig. 1

Comparison of IHC, FISH and qRT-PCR in ROS1 rearrangement and non-rearrangement cases (a–d). The H&E staining, IHC, FISH and qRT-PCR results of a non-rearrangement case. a The case presented acinar and papillary patterns in H&E staining (200×); b IHC showed no staining in tumor cells (200×); c, d it was confirmed by FISH and qRT-PCR as non-rearrangement, respectively; e–h A case with weak and focal ROS1 IHC staining. e The case presented acinar pattern in H&E staining (200×); f IHC showed weak to moderate focally staining in about 40 % tumor cells (200×); g, h it was also confirmed as non-rearrangement by FISH and qRT-PCR, respectively; i–l A case with diffusely moderate IHC staining. i The case presented papillary and micropapillary patterns in H&E staining (200×); j IHC showed moderate staining with cytoplasmic and focal granular patterns in about 80 % tumor cells (200×); k and l it was proved as ROS1-rearrangement by FISH and qRT-PCR, respectively; m-p A case with diffusely strong IHC staining. m The case presented papillary and micropapillary pattern in H&E staining (200×); n IHC showed diffusely strong staining with cytoplasmic, membrane and granular patterns in about 90 % tumor cells (200×); o, p it was also proved as ROS1 rearrangement by FISH and qRT-PCR

Fig. 2

The routine protocol for ROS1 arrangements detection in NSCLC. IHC with D4D6 mAb can be preliminary screening tool for ROS1 rearrangement detection in NSCLC, and the IHC cutoff value should be set at 2–3+ cytoplasmic reactivity with diffuse (≥60 % of the tumor cells) distribution or an H-score ≥150; FISH and qRT-PCR should be used as the secondary confirmation in the cases with weak of focal IHC staining